This application is the US National Stage Filing of PCR/FR98/02639 field on Dec. 7, 1998.
The present invention relates to a device intended for the qualitative or quantitative dosing of at least one particular component in a product-sample, preferably making it possible to implement immunological dosing, as well as to a process and a kit which are intended for implementing the device.
Numerous methods have been developed for identifying, detecting or quantifying analytes in chemical or biological samples.
These methods are mostly based on the formation of complexes by affinity reaction between members of a specific binding pair.
These reactions, of ligand/receptor type, result for example from interactions between an antigen and a specific antibody, from hybridization between two complementary nucleic acid sequences or from a phenomenon of recognition between the binding site of a protein, for example an enzyme, hormone or other biological entity, and its ligand, substrate or receptor.
The formation of an affinity complex makes it possible to reveal the presence of the sought-after analyte in the sample. This analyte may possibly be quantified, if it is possible to separate the complexed forms from those remaining in the free state, or to measure the degree of occupancy of the specific ligands of the analyte.
This type of method of detecting and quantifying an analyte present in a sample, sometimes in trace amounts, is of great interest to research or analysis laboratories, especially clinical or biological analysis laboratories.
However, for routine use, the methods must be able to be applied simultaneously to a large number of samples. Furthermore, for one and the same sample, it is often necessary to carry out several tests.
Therefore, in most cases, the manual protocols of routine analysis involve several reactions and successive manipulation steps. These multiple tests are carried out on samples in series, in very large centers in which several tens of thousands of samplesxe2x80x94may be tested per day. Such multiple tests may therefore impose constraints and require relatively lengthy performance times. Additionally, the successive manipulations which they require may give rise to errors in the results.
The problem of the automation of this type of test therefore rapidly arises, and various devices have thus been devised in order to achieve automation, or, at the very least, a simplification of the successive steps indicated above.
These devices remain however for the most part relatively complex or adapted to the detection of a particular type of analyte (cell or molecule) or else only allow qualitative analyses to be carried out. Such devices are described in particular in the documents EP 0339277 and EP 0426729.
In particular, the document EP 339 277 describes a device for performing successive analytical reactions for dosing an analyte in a liquid assay sample involving analytical reactions between the analyte and analytical reagents which interact with the analyte to produce an analyte-dependent detectable response.
This device comprises a closed receptacle having a horizontal axis of rotation. This closed receptacle is delimited externally by a cylindrical wall and internally comprises two concentric spoon-shaped walls which define between themselves a sample inlet zone. Between said spoon-shaped walls and the cylindrical peripheral wall there are defined several reaction zones into which the specific analytical reagents are incorporated.
According to this document, the sample is introduced by an entry pathway into the inlet chamber defined between the spoon-shaped walls and opening toward the reaction zones. By pivoting said receptacle in a swinging movement about its horizontal axis, the liquid sample is transported by gravity into the reaction zones where it interacts with the reagents and then transported to an examination zone situated at the center of the receptacle.
Such a device is designed chiefly to avoid any centrifugation of the product when carrying out dosing.
The document JP HEI-5 215 750 also discloses a device for detecting and analyzing cellular populations, which comprises a horizontal circular dish mounted rotatably about a vertical axis. This open dish is covered with antibodies.
It is rotated about its vertical axis so that the sample introduced at its center is distributed under the action of a centrifugal force over said dish.
The subsequent washing steps are carried out in the same way by introducing the rinsing liquid at the center of the dish, the liquid being discharged toward the periphery of the dish during the rotation of the latter, while rinsing the surface on which the component to be dosed is fixed. In order to recover the rinsing liquid, there is provided a receptacle placed beneath the dish.
Finally, the document WO 94 25 159 discloses a device for the qualitative and/or quantitative dosing of a particular component in a sample of products, which comprises a substantially circular container mounted rotatably on a shaft for driving via a central housing, and in which test chambers are made which extend along radii of the container and which have a density gradient. In the central part of the container, there is provided an annular centrifugation chamber which is in communication with each test chamber.
This annular centrifugation chamber can be divided into two parts which communicate with each other via an upper opening.
The wall delimiting the first part of the centrifugation chamber is inclined so that the products are transferred from the first part to the second part by overflowing from the delimiting wall, via the communication orifice. Likewise, the wall delimiting the second part of the centrifugation chamber exhibits an inclined slope so that the mixture is transferred into each test chamber by overflowing via the communication orifice between the second part of the centrifugation chamber and said test chambers.
The slope of the wall delimiting the second part of the centrifugation chamber is greater than the slope of the wall delimiting the first part so that during centrifugation, the product passes firstly from the first part of the centrifugation chamber situated close to the axis of rotation of the container, to the second part of the centrifugation chamber before being transferred to the test chambers.
The invention proposes a novel arrangement of a dosing device which is of simple design and easy to use and which can be manipulated individually with a minimum number of manipulations so as to carry out dosing, allowing dosings to be carried out in proximity to the place of withdrawal of the product sample containing the particular component to be dosed, such a device exhibiting an optimized arrangement and furthermore making it possible to carry out assays in series from small quantities of samples.
More particularly, according to the invention, there is proposed a device for the qualitative and/or quantitative dosing of at least one particular component in a product sample by labeling and fixing, said device comprising a container and a cover which are assembled to form a closed receptacle.
This device is characterized in that said closed receptacle has a vertical axis, in that the container and the cover carry coaxial cylindrical walls which, while the container and cover are being assembled, position themselves pairwise one against the other thereby delimiting at least three concentric annular chambers inside the receptacle, namely from the axis an inlet chamber intended for receiving the sample and as appropriate allowing the labeling of the component, a chamber for fixing and reading said labeled component and a discharge chamber, in that the coaxial cylindrical walls forming separations between the successive annular chambers each comprise at least one opening, and in that the assembled cover and container are able to turn with respect to one another about the vertical axis and the openings of the coaxial cylindrical walls of the container and of the cover are placed at determined angular positions, in such a way that by displacement of one with respect to the other of the cylindrical walls of each pair, the openings of each pair of walls are able to be positioned opposite one another or in an angularly offset manner, so as to put into communication or isolate from one another the successive annular chambers.
According to a preferred arrangement of the device in accordance with the invention, said openings provided in the coaxial cylindrical walls of the container and of the cover are positioned in such a way that the openings of a pair of cylindrical walls are opposite one another so as to put into communication two successive annular chambers, the openings of the other pairs of cylindrical walls are positioned in an angularly offset manner so that the other annular chambers are isolated.
According to other advantageous characteristics of the device in accordance with the invention, on the cover and on the container if comprises means of indexed positioning of these latter. The cover comprises a nipple on the external face of one of these coaxial cylindrical walls which are situated outside the other cylindrical walls so as to form the external peripheral edge of the receptacle, said nipple forming a grip or a sill for turning the cover about the vertical axis with respect to the container. The device according to the invention comprises a central through-orifice isolated from the immediately adjacent annular chamber and intended to be threaded onto a vertical shaft for rotational driving for setting said receptacle into rotation.
In the bottom of the cover or in the bottom of the container, there may be provided an entry orifice to the inlet chamber.
According to a variant of the device in accordance with the invention, provision may be made for the receptacle to comprise several concentric fixing and reading chambers between the inlet chamber and the discharge chamber.
The device according to the invention exhibits an optimized ergonomic shape. More particularly, its receptacle exhibits the shape of a disk.
Advantageously, the container of the device according to the invention is made from a transparent material so as to allow reading of the labeled fixed components in the fixing and reading chamber through the walls of said receptacle. The cover may be opacified or be treated in such a way as to avoid parasitic radiations, it being possible for the reading to be performed with the aid of a CCD camera.
The device according to the present invention preferably comprises in the fixing chamber at least one receptor of the component to be dosed, said receptor being fixed in the chamber. It should be understood according to the present invention that the terms receptor and ligand will be used to designate in a generic manner two elements which are bound by strong interactions, and it can therefore relate equally to an antigen/antibody pair or to a nucleic acid/complementary nucleic acid pair or else to a true ligand and receptor or other strong interactions.
The techniques making it possible to fix proteins (antigens, antibodies for example) or nucleic acids on plastic or even glass surfaces are well known to the person skilled in the art, these dealing with technologies currently used in particular to fix components of this same type in the standard microtiter plates which are used for example in ELISA or else which may be adapted as a function of the type of polymer involved.
Preferably the receptor will be fixed on the bottom of the container, if possible in the form of a monolayer so as to allow easier reading. Indeed, when the means of reading is a CCD camera, the radiation will pass through the bottom of the container and will or will not be modified by the presence of a labeled component and will then be recovered after a second pass through the bottom of the container.
The technique involved is in a general manner akin to so-called sandwich immunological dosing processes, that is to say the element to be detected reacts with the receptor for example the antibody and is itself labeled by another element recognizing it, which carries either a physical label, that is to say particles, or else a chemical label for example with the aid of fluorescent elements or ones which may be rendered fluorescent.
In this regard, according to an advantageous characteristic of the invention, the labeling particles exhibit a sufficient diameter, preferably greater than or equal to around 100 times the diameter of the component(s) to be dosed and possess optical properties allowing their detection by counting.
The expression xe2x80x9cpossess optical propertiesxe2x80x9d is understood to mean the fact that said particles are able to reflect all or some of a luminous radiation emitted by a detection system, heading for said particles.
Although it is possible to label the component before introducing it into the device, the labeling element capable of labeling the component to be dosed will preferably be placed in the inlet chamber, for example in unfixed dry form, this involving for example labeled antibodies recognizing one of the epitopes of the antigen to be dosed, another antibody being fixed in the fixing and labeling chamber.
In the device according to the present invention, the fixed receptor is chosen as indicated above from among:
antibodies, antigens, complementary nucleic acid sequences,
true receptors for specifically dosing components which are:
antigens, antibodies, nucleic acid sequences, ligands of said receptors.
For the immunological dosings, use will preferably be made of an antigen or a labeled antibody to respectively dose the complementary element and another complementary element will be fixed in the fixing and reading chamber.
It is also possible to provide for multiple dosing making it possible to dose perhaps several antigens or several antibodies. To do this it is sufficient for the fixing and reading chamber to be divided into plurality of angular sectors on which mutually different receptors are fixed, each intended for fixing and reading a different labeled component. For this purpose, it may be particularly advantageous according to the invention for an angular sector of the fixing chamber to be left devoid of receptors so as to constitute a blank sector intended for carrying out an initialization reading of said device to zero it.
Reading may be carried out according to the invention by virtue of a CCD camera which may of course be controlled by a computerized device which will make it possible to reconstruct the dosing of each of the elements as a function of the readings which will be made on the various sectors.
According to a particularly advantageous characteristic of the device according to the invention, the CCD camera is able to count in a discretionary manner by emission/reception of a light signal, the number of labeled fixed components in each fixing and reading chamber, so as to obtain a digital detection signal.
This entails using particles or microspheres to label the component or components to be dosed in the sample, these particles preferably exhibiting a size greater than around 100 times the size of the sought-after molecules, and being capable of returning all or some of a luminous radiation emitted in their direction, reflected radiation constituting so many events which are captured by the CCD camera used and transmitted to a computerized tool adapted for expressing the detected events in terms of absolute number in real time.
The invention also proposes a process for the qualitative and/or quantitative dosing of at least one particular component in a product sample by labeling and fixing.
This process is characterized in that use is made of at least one device according to the invention which contains specific receptors for the component to be dosed, which are fixed in each fixing and reading chamber and in which process,
a) the product sample containing the labeled component is placed in the inlet chamber isolated from the other annular chambers,
b) the cover is turned with respect to the container in such a way as to put the inlet chamber into communication with each fixing and reading chamber, the discharge chamber being isolated from the other annular chambers,
c) the device is rotated about its vertical axis in such a way as to disperse by centrifugation in each fixing and reading chamber the product sample containing the labeled component, the latter then binding by strong interaction to the fixed specific receptors in each fixing and reading chamber,
d) the cover is turned with respect to the container in such a way as to put the fixing and reading chamber into communication with the discharge chamber,
e) the device is rotated about its axis in such a way as to disperse by centrifugation the surplus sample into the discharge chamber,
f) the inside of the device is rinsed with the aid of a rinsing liquid which is circulated by centrifugation through the various annular chambers of said device while reproducing the preceding steps b), c), d) and e) in such a way as to retain in each fixing and reading chamber only the labeled component bound by strong interaction to the fixed receptors,
g) said labeled component is detected and dosed through the wall(s) of said device.
The process according to the invention can be automated and is xe2x80x9cgenericxe2x80x9d, it can be applied to the detection and to the counting of target substances, either of a molecular, particulate, vesicular or cellular nature.
The invention also proposes an apparatus for implementing the aforesaid process, characterized in that it comprises a vertical shaft for rotational driving on which are threaded devices according to the invention, means for maintaining said devices distanced from one another, means for the bidirectional rotational driving of said vertical drive shaft, means for injecting product samples and rinsing liquid into the inlet chambers of said devices threaded on the drive shaft, and means for turning the covers of the devices relative to the containers in such a way as to put the various successive annular chambers of these containers into communication or to isolate them and a means for reading the labeled fixed agents.
Thus, by virtue of the apparatus according to the invention, it is advantageously possible to carry out automatically, according to the process in accordance with the invention, using the device according to the invention, dosings of various components of one and the same sample, or one and the same component in particular in several samples of different products.